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Title: Applications of coherent anti-Stokes Raman scattering (CARS) microscopy to cell biology
Author: Karuna, Arnica
ISNI:       0000 0004 5922 8608
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2016
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Traditionally, many advances in the field of biology have been driven by optical microscopy based techniques which reveal morphological information about the samples under study [1, 2, 3, 4]. The scope of the applications of these methods is limited due to the lack of contrast from most biological materials (cells and tissues) which are transparent to visible light. The introduction of extraneous materials (such as fluorescent quantum dots or other fluorescent proteins/labels) with affinity towards certain sub-cellular components which are then imaged, has emerged as a popular and powerful method to image biological materials. Fluorescence microscopy using visible wavelengths in its simplest application includes the identification and imaging of interesting features of a sample which are fluorescently labelled in a structurally/ chemically specific way. In more recent developments, fluorescence lifetimes have been imaged. Fluorescence based techniques can also be applied to track protein dynamics or drug delivery in live samples. Despite the benefits of imaging samples with labels, and a host of associated applications, the issues of photobleaching and induced phototoxicity remain. Another very important aspect which gains relevance in live sample imaging is that the system dynamics may be influenced by the introduction of fluorescent labels. Spectroscopy techniques, which rely on the material resonances are chemically specific, sensitive, and if combined with microscopy, bridge the gap between non-invasive imaging and fluorescence microscopy. Instead of an extraneous label, the contrast generated originates from molecular transitions of the chemical species in the sample. Coherent Raman scattering (CRS) based techniques rely on the chemical contrast generated due to molecular vibrations and have been applied to biology [5]. One CRS technique, stimulated Raman scattering (SRS) has been used to distinguish between the macromolecular constituents of cells [6, 7] and tissues [8]. Additionally, quantitative hyperspectral SRS has been demonstrated in polymer and lipid mixtures [6]. Another type of CRS, coherent anti-Stokes Raman scattering (CARS) [9] was reported in 1965, nearly half a decade before SRS. However, due to difficulties in implementation, CARS was not readily put to application. Since its revival in 1999 [10], CARS has emerged as a label-free, chemically specific microscopy technique and has been applied to image various biological materials [11, 12]. In addition to the studies of lipid rich samples [13, 14], spectral differences between the cytosol and the nucleus have been reported using CARS microscopy [15, 16]. However, none of the previous works in this field present full 3D hyperspectral data, or make quantitative volumetric estimates of the various chemical components present in the sample. With respect to biomedical/biochemical application based studies, literature suffers from a paucity of examples investigating the effects of drugs on biological materials using CARS microscopy. This project aims to overcome these shortcomings. In this work, CARS microscopy is applied to single cells (osteosarcoma, U-2OS which are lipid poor due to their functional profile) with volumetric quantitative analysis to determine the absolute masses of the component species. For the first time in our knowledge, full 3D hyperspectral data has been acquired and analyzed. Correlative fluorescence imaging to ascertain the origin of various components of the cells as imaged with CARS was also performed. Furthermore, reports of no observed (with CARS) correlation in protein content in the intranuclear region with the mitotic stage in cells one publication [16] have been disproved, shown in this thesis. Osteosarcoma is a rare type of cancer, most commonly diagnosed in children and adolescents. However, due to its rarity, it is not well researched. The usual line of treatment includes surgery followed by chemotherapy, of which Taxol (microtubule stabilizer) and ICRF-193 (topoisomerase II poison) form an important part. The effects of these drugs on cells are often investigated in literature using a range of techniques, of which, the most non-invasive one is chemically non-specific optical microscopy [17, 18]. Among the chemically specific methods used to perform such studies, flow cytometry [19, 20, 21] is one of the most commonly employed; and the most invasive methods, also in widespread use, are Western blotting and gel electrophoresis [22]. This means that in the best case scenario, we can perform optical microscopy on the cells with no chemical specificity or sensitivity, or sacrifice non-invasiveness for chemical information. Identifying a need for label-free methods to study the effects of these drugs, we applied CARS microscopy to study the effects of Taxol and ICRF-193 on U-2OS cells. This was done to determine whether CARS microscopy is suitable for population phenotyping and profiling the effects of anticancer drugs over a period of time, following treatment. Also for the first time, in this project, CARS microscopy has been demonstrated with chemical specificity and sensitivity on structurally and functionally multicellular 3D assemblies, organoids. In the past, other groups have reported studies on organoids, their metabolism and drug interactions using fluorescence microscopy [23, 24, 25], with the already mentioned shortcomings and pitfalls of photodamage and photobleaching. This thesis is structured into five chapters. The required background is given in the first two chapters. The first chapter introduces optical spectroscopy with emphasis on CARS, including a discussion of the theory and the various implementations of CARS microscopy. The second chapter contains the biology background in cells and cell division requisite for this project. An overview of the current state of the art in imaging techniques is also given. The set-up and analysis techniques used to acquire and analyze the data presented in this work are described in the third chapter, along with a characterization of the effects of the imaging optics and the sample’s refractive index on the analysis method and the quantitative calculations, using polystyrene and polymethymethacrylate beads of different sizes. The next two chapters describe the applications of CARS microscopy to fixed U- 2OS cells and organoids. In chapter four, the results of CARS imaging and spectral analysis of U-2OS cells correlated with two-photon fluorescence are shown. Furthermore, applications of CARS to study the effects of two kinds of anti-cancer drugs i.e, Taxol and ICRF-193 on U-2OS cells are demonstrated. Additionally, a side project not related to CARS microscopy, but presenting a simple method to quantitatively investigate the number of eGFP molecules attached to cyc-B, across the cell cycle is also described in this chapter. Chapter five demonstrates the suitability of CARS microscopy to image higher levels of biological organization, specifically organoids which are miniature lab grown 3D models of organs. The summary and outlook of this project are given in the last chapter, which is followed by the Appendices including additional detailed information referenced in the thesis. All 3D data are available as videos in the data DOI related to this thesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QC Physics